Microbial detection apparatus, microbial detection method, and sample container used therein

ABSTRACT

The sample container has a two-layer membrane filter comprising a first layer as an upper layer serving as a hydrophilic membrane filter and a hydrophobic membrane filter as an underlying second layer capable of filtering an aqueous solution without the use of a wetting agent and by means of a formed negative pressure. Using this sample container, a large amount of an aqueous sample solution is filtered by means of a negative pressure formed by a suction portion to capture microbes in the aqueous sample solution by the hydrophilic membrane filter. Then, the negative pressure is restored to normal pressure, and a microbial dissolution solution is then added to the membrane filter to retain the microbial dissolution solution for a given time on the hydrophobic membrane filter. Then, the microbial dissolution solution is dispensed to a reaction container containing a luminescent reagent, and luminescence is detected to detect the microbes.

CLAIM OF PRIORITY

The present application claims priority from Japanese patent applicationJP 2009-044648 filed on Feb. 26, 2009, the content of which is herebyincorporated by reference into this application.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a microbial detection apparatus, amicrobial detection method, and a sample container used therein.

2. Background Art

The detection of microbes or the determination of microbial counts ispracticed, for example, in pharmaceutical or food factories orregenerative medical facilities, as management for safety and healththat stops the microbial contamination of products before it starts. Inparticular, the determination of microbial counts is conducted under theprovisions of the Japanese Pharmacopoeia on products or raw materials inpharmaceutical factories as well as on air, the surface of walls orworkers' gloves, etc., within pharmaceutical factories. Thedetermination of microbial counts is also conducted in food factorieswith the introduction of HACCP (Hazard Analysis and Critical ControlPoint) and conducted as inspection of the food factories themselves ortheir working process on food products as well as on the surface ofwalls, floors, or cookware such as cutting boards and kitchen knifes.

The determination of microbial counts is generally performed by aculture method. The culture method is a microbial-count determinationmethod which comprises: for liquid samples, directly plating the samplesonto an agar plate medium or for samples in a state other than liquid,plating a liquid containing microbes washed out of the samples, onto amedium; culturing the microbes on the medium; and counting the number offormed colonies by use of the fact that one microbe forms one colony.

Another general approach is a membrane filter (MF) method by whichmicrobial counts are determined using a membrane filter. The MF methodis a determination method which comprises: filtering samples through amembrane filter to capture microbes; placing the membrane filter havingthe captured microbes, onto an agar plate medium and culturing themicrobes thereon; and counting the number of colonies. Alternatively, amethod without the use of culture comprises: spraying a mist of an ATPextraction reagent for extracting adenosine triphosphate (ATP) fromwithin microbes and a luminescent reagent containing luciferase andluciferin, onto a membrane filter having the captured microbes;photographing, using a CCD (charge coupled device) camera, the brightpoints of luminescence generated through the luciferase-luciferinreaction with ATP; and determining microbial counts (Japanese PatentApplication No. 9-317792).

SUMMARY OF THE INVENTION

Conventional culture methods involve plating microbe-containing samplesonto an agar plate medium and therefore have the problem of the highestsample amount being limited to approximately 1 mL. Accordingly, themethods have difficulty in determining the microbial counts for a largeamount of samples containing a small number of microbes. Also, themethods disadvantageously require a 24- to 72-hour culture to obtaindetermination results. As a result, pharmaceutical or food products,live cells, and the like must be shipped only after the obtainment ofdetermination results. This gives rise to a large loss of safety,efficiency, and economy. Thus, it has been demanded to reduce the timerequired for the determination of microbial counts.

In conventional MF methods, nutrients in a medium may hardly bedistributed to microbes if intimate contact is not completely providedbetween a membrane filter having the captured microbes and the medium,due to air entering therebetween. In such a case, the growth rates ofthe microbes are not constant, disadvantageously resulting in varyingsizes of colonies and reduced determination precision. The MF methodsinvolve spraying an ATP extraction reagent onto microbes captured in themembrane filter. If the sprayed extraction reagent is diluted withsamples or buffer solutions infiltrated in the membrane filter or if themicrobial cell walls are strong enough to resist the given amount of theextraction reagent, extraction efficiency is reduced, resulting inreduced determination sensitivity.

An object of the present invention is to rapidly, highly precisely,highly sensitively, and conveniently detect and determine a small numberof microbes contained in a large amount of a sample.

We have intended to provide an apparatus and a method for rapidly,highly precisely, highly sensitively, and conveniently detecting anddetermining a small number of microbes contained in a large amount of asample, and have therefore attempted to develop an apparatus and amethod by which a microbe-containing sample is filtered through amembrane filter; the microbes are dissolved in a sufficient amount of amicrobial dissolution solution; and the microbes are detected based onluminescent or fluorescent reaction with microbe-derived biologicalmaterials as an index. During the course of this process, we have firstfound the problems shown below.

When the microbe-containing sample is filtered through a poroushydrophilic membrane filter, microbes having a diameter evidently largerthan the pore size of the membrane filter are captured on the surface ofthe membrane filter. By contrast, microbes having a diameter close tothe pore size of the membrane filter have been found to infiltrate intothe membrane filter and be thereby captured therein. When a microbialdissolution reagent (e.g., ATP extraction reagent) is added onto themembrane filter having the microbes thus captured therewithin, themicrobes have been found to be dissolved within the membrane filter suchthat most of the extracted biological materials (e.g., ATP) remain inthe membrane filter. When the extracted ATP on the membrane filter istransferred therefrom together with the ATP extraction reagent in such astate to a container containing a bioluminescent reagent (e.g.,luciferase/luciferin) and subjected to luminescence measurement,luminescence intensity has been found to be decreased due to ATPremaining as a loss in the membrane filter, resulting in reduceddetermination performance.

Thus, we have also attempted a method which comprises adding theextraction reagent onto the hydrophilic membrane filter and leaving itstanding or stirring the solution for a given time to liberate ATP fromwithin the membrane filter. However, in such a case, ATP has been foundto penetrate through the membrane filter and drop off, together with theextraction reagent, before being liberated from within the membranefilter, resulting in difficult liberation.

We have further attempted a method using a hydrophobic membrane filterimpermeable to aqueous solutions, instead of a hydrophilic membranefilter, to prevent the ATP extraction reagent from penetrating throughthe membrane filter and dropping off. First, the hydrophobic membranefilter must be infiltrated with a wetting agent (e.g., alcohols such asmethyl alcohol and ethyl alcohol or ethers such as diethyl ether) forfiltering a microbe-containing aqueous sample solution through thehydrophobic membrane filter. However, the wetting agent dissolves ordamages microbes. In addition, elution of the wetting agent from themembrane filter during filtration deteriorates filtration performance.Thus, this approach which involves infiltrating the hydrophobic membranefilter with a wetting agent prior to filtration has been found to bedifficult to use in microbial detection/quantification and microbialviability determination.

The present invention has been achieved to solve these problems found byus and the problems of the conventional techniques. We have found thateven using a hydrophobic membrane filter, an aqueous sample solution canbe filtered, depending on its pore size, without the use of a wettingagent by forming a negative pressure below the membrane filter, and havealso found that the extraction reagent neither penetrates through themembrane filter nor drops off when normal pressure is provided below thehydrophobic membrane filter. The filtration of an aqueous solutionthrough the hydrophobic membrane filter is achieved with a pore sizeequal to or larger than a certain size. Certain microbes having a sizesmaller than the pore size are hardly captured. Thus, we have developeda two-layer membrane filter which is capable of filtering a large amountof an aqueous solution, capturing microbes, and retaining a microbialdissolution solution, by providing, on the hydrophobic membrane filter,a porous hydrophilic membrane filter with a very small pore size forcapturing microbes.

A microbial detection apparatus of the present invention comprises: asample container comprising, in the bottom, a two-layer membrane filtercomprising a first layer as an upper layer serving as a hydrophilicmembrane filter and a hydrophobic membrane filter as an underlyingsecond layer capable of filtering an aqueous solution without the use ofa wetting agent and by means of a formed negative pressure; and asuction portion provided below the two-layer membrane filter in thesample container. The hydrophilic membrane filter as the first layer ofthe two-layer membrane filter is responsible for capturing microbes,while the hydrophobic membrane filter as the second layer is responsiblefor retaining a reagent.

According to the present invention, a large amount of an aqueous samplesolution can be filtered through the hydrophilic membrane filter as thefirst layer and the hydrophobic membrane filter as the second layer bymeans of a negative pressure formed by the suction portion. Thus,microbes in the aqueous sample solution can be captured by thehydrophilic membrane filter as the first layer without being dissolvedor damaged. Then, the negative pressure is restored to normal pressure,and a microbial dissolution solution is then added to the membranefilter. As a result, the hydrophobic membrane filter as the second layercan retain thereon the microbial dissolution solution for a given timeto prevent the microbial dissolution solution from penetrating throughthe membrane filter and dropping off. According to the presentinvention, the microbes captured by the hydrophilic membrane filter asthe first layer can be dissolved, and microbe-derived biologicalmaterials within the hydrophilic membrane filter as the first layer canbe liberated into the solution on the hydrophilic membrane filter as thefirst layer.

The hydrophilic membrane filter as the first layer has a pore size ofpreferably 0.05 μm to 0.65 μm. The hydrophobic membrane filter as thesecond layer has a pore size of preferably 0.8 μm to 80 μm. Thehydrophilic membrane filter as the first layer having a pore size set to0.05 μm to 0.65 μm can reliably capture microbes. The hydrophobicmembrane filter as the second layer having a pore size set to 0.8 μm to80 μm can filter an aqueous sample solution without the use of a wettingagent and by means of a formed negative pressure and can retain thereona microbial dissolution solution under normal pressure restored from thenegative pressure.

A microbial detection method of the present invention comprises: addinga first reagent for degrading an extra-microbial biological material, toan aqueous sample solution to degrade the extra-microbial biologicalmaterial; suction-filtering the aqueous sample solution through thetwo-layer membrane filter to capture microbes by the hydrophilicmembrane filter as the first layer while removing the first reagent andforeign substances contained in the aqueous sample solution; then addingthereto a second reagent for extracting biological materials within themicrobes, and retaining the second reagent on the hydrophobic membranefilter as the second layer to extract the biological materials from themicrobes and liberate the biological materials from within thehydrophilic membrane filter as the first layer; and allowing theliberated biological materials to act on a third reagent reactivethereto, followed by quantitative measurement of the microbes. In thiscontext, the second reagent used is a reagent that does not infiltrateinto the hydrophobic membrane filter.

The present invention provides a microbial detection apparatus and amicrobial detection method which are capable of easily filtering a largeamount of an aqueous sample solution, reliably dissolving microbes, andeasily eluting, from the membrane filter, biological materials extractedfrom the microbes. The present invention also provides a microbialdetection apparatus and a microbial detection method which achievehighly sensitive, highly precise, rapid, and convenient microbialviability determination and live cell quantification without the use ofa culture and without the need of experience.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram showing a partial cross section of amicrobial detection apparatus according to an embodiment of the presentinvention.

FIG. 2 is a flowchart showing procedures of a microbial detectionprocess.

FIG. 3 is a schematic diagram showing a partial cross section of amicrobial detection apparatus according to an embodiment of the presentinvention.

FIG. 4 is a schematic diagram showing a partial cross section of amicrobial detection apparatus according to an embodiment of the presentinvention.

FIG. 5 is a schematic diagram showing a partial cross section of amicrobial detection apparatus according to an embodiment of the presentinvention.

FIG. 6 is a schematic diagram showing a partial cross section of amicrobial detection apparatus according to an embodiment of the presentinvention.

FIG. 7 (7A and 7B) is a diagram showing an example of how to mount atwo-layer membrane filter into the bottom of a container.

FIG. 8 (8A and 8B) is a diagram showing an example of how to mount atwo-layer membrane filter into the bottom of a container.

FIG. 9 (9A to 9D) is a schematic diagram showing the intermediateprocess of a detection method using the two-layer membrane filteraccording to the present invention.

FIG. 10 (10A to 10D) is a schematic diagram showing the intermediateprocess of a detection method using a single-layer hydrophilic membranefilter.

FIG. 11 (11A and 11B) is a diagram illustrating suction filtration.

FIG. 12 is a diagram showing the relationship between the number of E.coli in the abscissa and the number of ATP obtained using the two-layermembrane filter.

FIG. 13 is a diagram showing the number of ATP detected under variousconditions.

FIG. 14A (14A-a to 14A-c) is a diagram illustrating a measurementexperiment on the ability of the two-layer membrane filter to retain amicrobial dissolution solution.

FIG. 14B (14B-a to 14B-c) is a diagram illustrating a measurementexperiment on the ability of a single-layer hydrophilic membrane filterto retain a microbial dissolution solution.

FIG. 15 (15A to 15D) is a diagram illustrating an elution experiment onATP molecules within the two-layer membrane filter.

FIG. 16 (16A to 16D) is a diagram illustrating an elution experiment onATP molecules within a single-layer hydrophilic membrane filter.

FIG. 17 is a diagram showing time-dependent change in the luminescenceintensity of a microbial dissolution solution that remained on asingle-layer hydrophilic membrane filter and a microbial dissolutionsolution that penetrated through the membrane filter and dropped off.

FIG. 18 is a diagram showing time-dependent change in the luminescenceintensity of a microbial dissolution solution on the two-layer membranefilter.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Hereinafter, the embodiments of the present invention will be describedwith reference to the drawings. However, the present invention is notlimited to the description below by any means.

Example 1

(Microbial Detection Apparatus and Method)

FIG. 1 is a schematic diagram showing a partial cross section of amicrobial detection apparatus according to an embodiment of the presentinvention. The microbial detection apparatus comprises a container 102comprising a two-layer membrane filter 101 in the bottom, a reagentsupply portion 103, a pipe 104 for reagent supply, a suction portion105, a washing solution supply portion 106, a pipe 107 for washingsolution supply, a heating portion 108, an arm 109, a dispensing portion110, a pipe 111 for dispensing, a detection portion 112, a reactioncontainer 113, and an input/control portion 114. The input/controlportion 114 performs centralized control of the operation of eachportion in the apparatus. The container 102 is detachable/attachablefrom/to the apparatus. The reaction container 113 contains a luminescentreagent 115.

The two-layer membrane filter 101 has a first layer as an upper layerserving as a hydrophilic membrane filter 116 and a second layer as alower layer serving as a hydrophobic membrane filter 117. Thehydrophilic membrane filter 116 as the first layer has a pore size setto 0.05 μm to 0.65 μm for reliably capturing microbes. It also has athickness set to 7 μm to 200 μm. Of course, the hydrophilic membranefilter 116 may have a thickness smaller or larger than this range.However, a membrane filter thinner than this range has weak mechanicalstrength and is presumably broken during filtration. Alternatively, amembrane filter thicker than this range presumably allows a large amountof a solution to remain therein, with which various reagents addedthereafter are diluted. Thus, the thickness is preferably 7 μm to 200μm. The hydrophobic membrane filter 117 as the second layer has a poresize set to 0.8 μm to 80 μm for reliably filtering an aqueous solution.It also has a thickness set to 7 μm to 800 μm. Of course, thehydrophobic membrane filter 117 may have a thickness smaller or largerthan this range. However, a membrane filter thinner than this range hasweak mechanical strength and is presumably broken during filtration.Alternatively, a membrane filter thicker than this range presumablyrequires time for suction filtration. Thus, the thickness is preferably7 μm to 800 μm.

FIG. 2 is a flowchart showing procedures of a microbial detectionprocess using the microbial detection apparatus shown in FIG. 1. FIGS. 3to 6 respectively show alternative embodiments of the microbialdetection apparatus of the present invention, which will be describedbelow together therewith.

The container 102 comprising the two-layer membrane filter 101 in thebottom is loaded in the apparatus. First, initial values as to the highor low viscosity of a sample or whether or not Bacillus subtilis in aspore form can be measured are input to the input/control portion 114,and microbial detection is initiated (S201). An aqueous sample solutioncontaining microbes to be measured is added to the container 102 (S202).When the aqueous sample solution is judged in step 203 as having highviscosity, the input/control portion 114 drives the heating portion 108to heat the aqueous sample solution for promoting quick filtration(S204). When the sample viscosity input in the step 201 is low, theprocess goes to step 205 without the heating step.

Next, an extra-microbial biological material degradation reagent issupplied from the reagent supply portion 103 through the pipe 104 forreagent supply to the aqueous sample solution in the container 102(S205). The extra-microbial biological material degradation reagent usedis, for example, ATPase, DNase (DNA: deoxyribonucleic acid), or RNase(RNA: ribonucleic acid).

As shown in FIG. 3, the microbial detection apparatus may comprise,instead of a reagent supply portion, a reagent reservoir 302 containingvarious reagents 301. In such a case, the microbial detection apparatusmay comprise a dispensing portion 110 which dispenses an extra-microbialbiological material degradation reagent in the reagent reservoir 302through the pipe 111 for dispensing to the aqueous sample solution. Theapparatus having the reagent supply portion 103 shown in FIG. 1 issuitable in uses for measuring a large number of samples per day, andthe apparatus having the reagent reservoir shown in FIG. 3 is suitablein uses for measuring a small number of samples.

Next, the aqueous sample solution in the container 102 is filteredthrough the two-layer membrane filter 101 by means of a negativepressure formed by the suction portion 105. In this filtration, microbesin the aqueous solution are captured by the hydrophilic membrane filteras the first layer, while the biological material degradation reagent orresidual biological materials flow into a waste liquid portion 118 forremoval. Then, a washing solution is supplied from the washing solutionsupply portion 106 for the purpose of washing the two-layer membranefilter 101 (S206). When the aqueous sample solution has high viscosity,a washing solution may be supplied from the washing solution supplyportion 106 through the pipe 107 for washing solution supply for thepurpose of reducing the viscosity. Likewise, for the purpose of reducingthe viscosity, the washing solution may be heated by the heating portion108 and then added to the aqueous sample solution.

Next, when the microbes to be measured are judged in step 207 as havingsporulated, a vegetative cell conversion reagent is added thereto fromthe reagent supply portion 103 after the filtration (S208). Thevegetative cell conversion reagent used is, for example, combination ofalanine, glucose, and phosphoric acid or may be a liquid medium. For themicrobial detection apparatus comprising the reservoir 302 for variousreagents shown in FIG. 3, a vegetative cell conversion reagent in thereagent reservoir 302 is dispensed by the dispensing portion 110 to theaqueous sample solution. The vegetative cell conversion reagent may alsobe heated by the heating portion 108 for promoting the conversion tovegetative cells (S209). Furthermore, the solution may be filtered forthe purpose of removing the vegetative cell conversion reagent (S210).When the judgment in the step 207 is No, the process goes to step 211without the procedures of the steps 208 to 210.

Next, a microbial dissolution solution is added from the reagent supplyportion 103 to retain the microbial dissolution solution on thetwo-layer membrane filter 101 (S211). The microbial dissolution solutionused is, for example, benzalkonium chloride, trichloroacetic acid, or aTris buffer. For the microbial detection apparatus comprising thereservoir 302 for various reagents shown in FIG. 3, a microbialdissolution solution in the reagent reservoir 302 is dispensed by thedispensing portion 110 to the aqueous sample solution.

Next, the microbial dissolution solution on the two-layer membranefilter 101 in the container 102 is aspirated through the pipe 111 fordispensing of the dispensing portion 110, and the arm 109 is driven tomove the pipe 111 for dispensing to above the reaction container 113 ofthe detection portion 112 such that the microbial dissolution solutionis transferred to the reaction container 113 (S212). The reactioncontainer 113 contains a luminescent reagent 115, which in turn reactswith biological materials (e.g., ATP, luminol, or alkaline phosphatase)contained in the microbial dissolution solution to generateluminescence. The luminescent reagent 115 used is, for example,luciferase/luciferin. Alternatively, substrates for peroxidase oralkaline phosphatase may be used. In this case, animal cells can also bedetected.

As in the embodiments shown in FIGS. 4 and 5, the microbial detectionapparatus may comprise the detection portion 112 on the side of or belowthe container 102. In such a case, luminescence may be generated bysupplying a luminescent reagent by the reagent supply portion 103directly onto the two-layer membrane filter 101 in the container 102. Inthe embodiment of the microbial detection apparatus shown in FIG. 5, acontainer used comprises a two-layer membrane filter 101 placed not allover the bottom but in the partial region thereof and has a transparentregion in the bottom. The detection portion 112 is disposed to face thetransparent region in the bottom of the container and detectsluminescence generated through luminescent reaction from the solution inthe container. Alternatively, as in the embodiment of the apparatusshown in FIG. 6, the microbial detection apparatus may comprise anexcitation light irradiation portion 601. In such a case, fluorescencemay be generated by irradiating biological materials (e.g., DNA, RNA, orNAD (nicotinamide adenine dinucleotide)) with excitation light from theexcitation light irradiation portion 601 without the use of aluminescent reagent.

The generated luminescence or fluorescence is detected by the detectionportion 112 (S213), and microbial counts are calculated based on thedetected luminescence or fluorescence intensity (S214) to completemicrobial detection.

When the process shown in FIG. 2 goes from the step 207 to the step 211without the procedures of the steps 208 to 210, followed by luminescencemeasurement in the step 213, the number of vegetative cells contained inthe sample can be detected. Alternatively, when the procedures from thesteps 208 to 214 are performed after the step 206, the number ofnon-vegetative cells can be detected. Thus, the numbers of vegetativecells and non-vegetative cells contained in the sample can bedifferentiated therebetween and detected by detecting the number ofvegetative cells by the process without the steps 208 to 210 and thenrepeating the procedures from the steps 208 to 214.

Example 2

(Microbial Detection Experiment 1: E. Coli)

E. coli detection sensitivity was compared between the containercomprising the two-layer membrane filter according to the presentinvention and a container comprising a conventional hydrophilic membranefilter.

Two containers equipped with a membrane filter were prepared, one ofwhich was the container 102 comprising the two-layer membrane filter 101in the bottom and the other of which was a container comprising only ahydrophilic membrane filter in the bottom.

Referring to FIG. 7 (7A and 7B), an example of how to mount thetwo-layer membrane filter into the bottom of the container will bedescribed. The two-layer membrane filter 101 comprises a first layer asan upper layer serving as a hydrophilic membrane filter 116 and a secondlayer as a lower layer serving as a hydrophobic membrane filter 117. Thehydrophilic membrane filter 116 used was an MF-Millipore membrane filter(Nihon Millipore Ltd.) having a pore size of 0.45 μm, a thickness of 150μm, and porosity of 79% and was processed into 0.5 cm in diameter. Thehydrophilic membrane filter may be a Durapore or Isopore membrane filter(Nihon Millipore Ltd.) instead. The hydrophobic membrane filter 117 usedwas a Mitex membrane filter (Nihon Millipore Ltd.) having a pore size of10 μm and was processed into 0.5 cm in diameter. The hydrophobicmembrane filter may be a polypropylene prefilter (Nihon Millipore Ltd.)having a pore size of 30 μm instead. Alternatively, the hydrophobicmembrane filter may be made of a material such as nylon,polytetrafluoroethylene, hydrophobic polyvinylidene fluoride,polyethylene, polysiloxane, polycarbonate, polysulfone, polyamide, orglass fiber and may be a membrane filter having a pore size of 0.8 μm to80 μm or a membrane filter having a mesh structure with a pitch size of1 μm to 59 μm.

As shown in FIG. 7A, the processed hydrophilic membrane filter 116 wasoverlaid on the processed hydrophobic membrane filter 117, and theresulting filter was mounted to a mounting portion provided in a bottom801 of the container 102. The outer edge of the filter was supported byan annular cap 802 and secured with a screw 803 as shown in FIG. 7B tocomplete assembly. Alternatively, the filter may be secured using ascrewed cap. For example, as shown in FIG. 8A, the processed hydrophilicmembrane filter 116 is overlaid on the processed hydrophobic membranefilter 117, and the resulting filter may be inserted between a screwedbottom 901 of the container 102 and a screwed annular cap 902 andsecured with the bottom 901 and the cap 902 as shown in FIG. 8B forassembly. In this context, a gap may be formed between the hydrophilicmembrane filter as the first layer and the hydrophobic membrane filteras the second layer.

The container comprising only the hydrophilic membrane filter, which hasthe same container structure thereas, was a container 1002 equipped witha single-layer hydrophilic membrane filter 1001 instead of a two-layermembrane filter, as shown in an abbreviated form in FIG. 10A. Thesingle-layer hydrophilic membrane filter 1001 used was an MF-Milliporemembrane filter (Nihon Millipore Ltd.) having a pore size of 0.45 μm, athickness of 150 μm, and porosity of 79% and was processed into 0.5 cmin diameter.

FIG. 9 (9A to 9D) is a schematic diagram showing the intermediateprocess of a detection method using the container comprising thetwo-layer membrane filter according to the present invention. FIG. 10(10A to 10D) is a schematic diagram showing the intermediate process ofa detection method using the container comprising the single-layerhydrophilic membrane filter.

E. coli was used as the microbe to be measured. E. coli 701 wassuspended in a phosphate buffer (pH 7.4) (Invitrogen Corp.) foradjusting the number of E. coli to 20 to 2000 individuals/10 mL toprepare an E. coli suspension 702.

As shown in FIGS. 9A and 10A, 10 mL of the E. coli suspension 702 wasfirst added onto each of the two-layer membrane filter 101 and thesingle-layer hydrophilic membrane filter 1001. Next, 10 μL of an ATPelimination solution included in Lucifer HS Set (Kikkoman Corp.) wasadded thereto as an extra-microbial biological material removal reagent.Subsequently, as shown in FIG. 11A, a suction port 1101 of the suctionportion 105 was connected to below the two-layer membrane filter. Asshown in FIG. 11B, a negative pressure is formed to filter the E. colisuspension and the ATP elimination solution. Likewise, through thesingle-layer hydrophilic membrane filter, the E. coli suspension and theATP elimination solution were filtered. FIGS. 9B and 10B respectivelyshow a state after the filtration. After the filtration, 200 μL of anATP extraction solution included in the Lucifer HS Set (Kikkoman Corp.)was added thereto as a microbial dissolution solution 703 to extract ATPmolecules 704 from the E. coli 701, as shown in FIGS. 9C and 10C.

While left standing, the microbial dissolution solution 1003 penetratedthrough the single-layer hydrophilic membrane filter 1001 and droppedoff as shown in FIG. 10D. On the other hand, no change was observed inthe two-layer membrane filter 101 as shown in FIG. 9D.

10 μL of the microbial dissolution solution 703 remaining on each of thetwo-layer membrane filter 101 and the single-layer hydrophilic membranefilter 1001 was dispensed using the dispensing portion 110 to aluminescent reagent 115 (included in the Lucifer HS set (KikkomanCorp.)) contained in the reaction container 113 placed on the detectionportion 112. The luminescence intensity was calculated as the number ofATP. The results obtained using the two-layer membrane filter 101 wereplotted in a diagram with the number of ATP as the ordinate against thenumber of E. coli as the abscissa, as shown in FIG. 12.

When the container comprising the two-layer membrane filter was used,the number of ATP and the number of E. coli exhibited a quantitativelinear relation (y=1.4315x−7.9823) in which the number of ATP moleculeswas 1.4 amol on average per E. coli. By contrast, when the containercomprising the single-layer hydrophilic membrane filter was used, ATPmolecules could not be detected from less than 100 individuals of E.coli, and the number of ATP was 1 amol per 100 individuals of E. coli.Thus, the two-layer membrane filter 101 exhibited a value about 100times higher than that of the single-layer hydrophilic membrane filter1001.

This result may be because in the container comprising the single-layerhydrophilic membrane filter, the single-layer hydrophilic membranefilter cannot retain thereon the microbial dissolution solution, andtherefore, the microbial dissolution solution 1003 penetrates throughthe single-layer hydrophilic membrane filter and drops off together withE. coli-derived ATP molecules 1004. As a result, the E. coli-derived ATPmolecules 1004 were not eluted into the microbial dissolution solutionon the single-layer hydrophilic membrane filter 1001 (FIG. 10D).

On the other hand, in the container comprising the two-layer membranefilter, the two-layer membrane filter 101 can retain thereon themicrobial dissolution solution 703, and therefore, the microbialdissolution solution 703 neither penetrates through the two-layermembrane filter 101 nor drops off together with the E. coli-derived ATPmolecules 704. Moreover, the ATP molecules 704 extracted from E. coliwithin the hydrophilic membrane filter 116 as the first layer are elutedwith time into the microbial dissolution solution on the two-layermembrane filter 101 (FIG. 9D).

The microbial detection apparatus of the present invention could measureone E. coli highly sensitively, highly precisely, rapidly, andconveniently. The same effect is also obtained for bacteria such as acoliform group and Staphylococcus, yeast such as Saccharomycescerevisiae, and fungi such as Aspergillus niger.

Example 3

(Microbial Detection Experiment 2: Bacillus subtilis in Spore Form)

Detection sensitivity of Bacillus subtilis in a spore form was comparedbetween the container comprising the two-layer membrane filter accordingto the present invention and a container comprising a conventionalhydrophilic membrane filter.

Two containers equipped with a membrane filter were prepared, one ofwhich was the container 102 comprising the two-layer membrane filter 101and the other of which was a container comprising only a hydrophilicmembrane filter. The container structures and the structures in whichthe two-layer membrane filter or the single-layer hydrophilic membranefilter is mounted on the container are as described in FIGS. 7 and 8 (8Aand 8B).

The two-layer membrane filter 101 comprised a first layer as an upperlayer serving as a hydrophilic membrane filter 116 and a second layer asa lower layer serving as a hydrophobic membrane filter 117. Thehydrophilic membrane filter 116 used was an MF-Millipore membrane filter(Nihon Millipore Ltd.) having a pore size of 0.45 μm, a thickness of 150μm, and porosity of 79% and was processed into 0.5 cm in diameter. Thehydrophilic membrane filter may be a Durapore or Isopore membrane filter(Nihon Millipore Ltd.) instead. The hydrophobic membrane filter 117 usedwas a Mitex membrane filter (Nihon Millipore Ltd.) having a pore size of10 μm and was processed into 0.5 cm in diameter. The hydrophobicmembrane filter may be a polypropylene prefilter (Nihon Millipore Ltd.)having a pore size of 30 μm instead. Alternatively, the hydrophobicmembrane filter may be made of a material such as nylon,polytetrafluoroethylene, hydrophobic polyvinylidene fluoride,polyethylene, polysiloxane, polycarbonate, polysulfone, polyamide, orglass fiber and may be a membrane filter having a pore size of 0.8 μm to80 μm or a membrane filter having a mesh structure with a pitch size of1 μm to 59 μm.

The single-layer hydrophilic membrane filter mounted in the containercomprising only the hydrophilic membrane filter was an MF-Milliporemembrane filter (Nihon Millipore Ltd.) having a pore size of 0.45 μm, athickness of 150 μm, and porosity of 79% and was processed into 0.5 cmin diameter.

Bacillus subtilis in a spore form was used as the microbe to bemeasured. Bacillus subtilis was suspended in a 10% w/v gelatinsolution+phosphate buffer (pH 7.4) (Invitrogen Corp.) to prepare ahighly viscous Bacillus subtilis suspension having 2000 individuals ofBacillus subtilis/10 mL. 100 mM alanin+100 mM glucose+phosphate buffer(pH 7.4) was used as a vegetative cell conversion reagent. An ATPelimination solution included in the Lucifer HS Set (Kikkoman Corp.) wasused as an extra-microbial biological material removal reagent. An ATPextraction solution included in the Lucifer HS Set (Kikkoman Corp.) wasused as a microbial dissolution solution.

The microbial detection apparatus shown in FIG. 3 was used in thismeasurement. In the present Example, a touch panel display was used forinput to the input/control portion 114. Alternatively, the input/controlportion 114 used may be, for example, a laptop computer, a desktopcomputer, or an input button combined with a display and a USB(universal serial bus) memory for input/output.

First, the Bacillus subtilis suspension is highly viscous because itcontains gelatin. The Bacillus subtilis suspension also containssporulated bacteria. Therefore, the information about the high viscosityof the Bacillus subtilis suspension and the sporulated bacteriacontained therein was input using the input/control portion 114.Subsequently, the input/control portion 114 directed the apparatus toinitiate microbial detection. The apparatus performs detection processaccording to the steps shown in FIG. 2.

10 mL of the Bacillus subtilis suspension was added onto each of thetwo-layer membrane filter and the single-layer hydrophilic membranefilter (S202). The input/control portion 114 judged the Bacillussubtilis suspension in step 203 as having high viscosity based on theinput information and controlled the heating portion 108 to heat theBacillus subtilis suspension at 40° C. for reducing the viscosity(S204).

10 μL of the ATP elimination solution was added to the Bacillus subtilissuspension (S205), and the Bacillus subtilis suspension and the ATPelimination solution were filtered through each of the two-layermembrane filter 101 and the single-layer hydrophilic membrane filter bymeans of a negative pressure formed by the suction portion 105 (S206).

After the filtration, the input/control portion 114 judged the solutionin step 207 as containing sporulated bacteria based on the inputinformation. For converting the spores to vegetative cells, 1 mL of thevegetative cell conversion reagent was added onto each of the two-layermembrane filter 101 and the single-layer hydrophilic membrane filter(S208). The vegetative cell conversion reagent was heated at 40° C. or45° C. for approximately 1 hour by the heating portion 108 to promotethe conversion of the spores to vegetative cells (S209). After 1 hour,the vegetative cell conversion reagent penetrated through thesingle-layer hydrophilic membrane filter and dropped off. By contrast,the vegetative cell conversion reagent was retained on the two-layermembrane filter 101 and therefore filtered again (S210).

Subsequently, 200 μl of the ATP extraction reagent was added thereto andleft standing for 10 minutes (S211). While left standing, the ATPextraction solution penetrated through the single-layer hydrophilicmembrane filter and dropped off. By contrast, no change was observed inthe two-layer membrane filter 101. 10 μL of the ATP extraction solutionremaining on each of the two-layer membrane filter 101 and thesingle-layer hydrophilic membrane filter was dispensed by the dispensingportion 110 to a luminescent reagent 115 contained in the reactioncontainer 113 placed on the detection portion 112 (S212). The detectionportion 112 detected generated luminescence (S213), and theinput/control portion 114 calculated the number of ATP from theluminescence intensity (S214).

The results of detecting the number of ATP are shown in FIG. 13. Whenthe single-layer hydrophilic membrane filter was used, the number of ATPwas approximately 10 amol per 100 individuals of Bacillus subtilis. Bycontrast, when the two-layer membrane filter 101 was used, the number ofATP was an estimate of approximately 200 amol, which was 20 times higherthan that of the single-layer hydrophilic membrane filter. This resultmay be because the single-layer hydrophilic membrane filter cannotretain thereon the ATP extraction solution, and therefore, the ATPextraction solution penetrates through the single-layer hydrophilicmembrane filter and drops off together with Bacillus subtilis-derivedATP molecules. As a result, the Bacillus subtilis-derived ATP moleculeswere not eluted into the solution on the single-layer hydrophilicmembrane filter. On the other hand, the two-layer membrane filter 101can retain thereon the ATP extraction solution, and therefore, the ATPextraction solution neither penetrates through the two-layer membranefilter 101 nor drops off together with the Bacillus subtilis-derived ATPmolecules. Thus, the Bacillus subtilis-derived ATP molecules within thehydrophilic membrane filter 116 as the first layer could be eluted withtime into the solution on the two-layer membrane filter 101.

When the vegetative cell conversion reagent was not heated by theheating portion 108 (25° C., room temperature in FIG. 13), the number ofATP was approximately 10 amol per 100 individuals of Bacillus subtilis.

Thus, the microbial detection apparatus of the present invention couldmeasure Bacillus subtilis highly sensitively, highly precisely, rapidly,and conveniently.

The present invention has been completed by the accumulation of variousexperiments. Hereinafter, the experiments on which the present inventionis based will be described.

Experimental Example 1

(Examination of Hydrophobic Membrane Filter Capable of Retaining AqueousSolution and Filtering the Aqueous Solution Under Negative Pressure)

The presence or absence of a hydrophobic membrane filter was examined,which is capable of retaining an aqueous solution and filtering theaqueous solution under negative pressure.

The pore sizes, pitch sizes, and materials of the examined hydrophobicmembrane filters are shown in Table 1. The aqueous solutions used in theexperiment are ultrapure water, a phosphate buffer, and a microbialdissolution solution. The phosphate buffer is 50 mM phosphoric acid/NaOHbuffer, pH 7.4. The microbial dissolution solution is 0.2% benzalkoniumchloride+25 mM Tricine buffer, pH 12.

TABLE 1 Pore Dropwise addition Filtration under negative pressure sizeMicrobial Microbial Material (μm) Water Phosphate dissolution solutionWater Phosphate dissolution solution Polytetrafluoroethylene 10 RetainedRetained Retained Possible Possible Possible 3 Retained RetainedRetained Possible Possible Possible 0.45 Retained Retained RetainedImpossible Impossible Impossible Polypropylene 80 Retained RetainedRetained Possible Possible Possible 30 Retained Retained RetainedPossible Possible Possible 0.6 Retained Retained Retained DifficultDifficult Difficult Polyvinylidene fluoride 0.45 Retained RetainedRetained Impossible Impossible Impossible 0.22 Retained RetainedRetained Impossible Impossible Impossible 0.1 Retained Retained RetainedImpossible Impossible Impossible Nylon 0.8 Retained Retained RetainedPossible Possible Possible Pitch Dropwise addition Filtration undernegative pressure size Microbial Microbial Material (μm) Water Phosphatedissolution solution Water Phosphate dissolution solution Nylon 59Retained Retained Retained Possible Possible Possible 38 RetainedRetained Retained Possible Possible Possible 25 Retained RetainedRetained Possible Possible Possible 10 Retained Retained RetainedPossible Possible Possible 5 Retained Retained Retained PossiblePossible Possible 1 Retained Retained Retained Possible PossiblePossible

The aqueous solutions were separately added dropwise onto eachhydrophobic membrane filter to examine the ability to retain the aqueoussolution (Table 1). As a result, all the aqueous solutions could beretained on the hydrophobic membrane filters made of any of thematerials and neither penetrated through the membrane filter nor droppedoff.

The hydrophobic membrane filters were also examined for whether or noteach aqueous solution could be filtered therethrough by means of anegative pressure (Table 1). As a result, the hydrophobic membranefilters having a pore size of 0.8 μm to 80 μm were impermeable to theaqueous solutions under normal pressure and could filter the aqueoussolutions under negative pressure. The hydrophobic membrane filtershaving a pore size of 0.6 μm or smaller hardly achieved filtration undernegative pressure, and the hydrophobic membrane filters having a poresize of 0.45 μm failed to filter the aqueous solutions. In addition, thehydrophobic membrane filters having a mesh structure with a pitch sizeof 1 μm to 59 μm retained the aqueous solutions and could filter themunder negative pressure.

Moreover, a hydrophilic membrane filter having a pore size of 0.05 μm to0.65 μm was overlaid onto a hydrophobic membrane filter having a poresize of 0.8 μm to 80 μm or a pitch size of 1 μm to 59 μm to prepare atwo-layer membrane filter. Filtration was attempted by adding aqueoussolutions onto the two-layer membrane filter. As a result, the two-layermembrane filter also achieved filtration of the aqueous solutions.

Experimental Example 2

(Measurement Experiment on Ability of Two-Layer Membrane Filter toRetain Microbial Dissolution Solution)

The ability to retain a microbial dissolution solution was comparedbetween the two-layer membrane filter and a single-layer hydrophilicmembrane filter. For this purpose, two containers comprising eithermembrane filter in the bottom were prepared.

One of the containers was the container 102 comprising the two-layermembrane filter 101 (FIG. 14A-a). The two-layer membrane filter 101comprised a first layer as an upper layer serving as a hydrophilicmembrane filter 116 and a second layer as a lower layer serving as ahydrophobic membrane filter 117. The hydrophilic membrane filter 116 asthe first layer used was an MF-Millipore membrane filter (NihonMillipore Ltd.) having a pore size of 0.45 μm, a thickness of 150 μm,and porosity of 79% and was processed into 0.5 cm in diameter. Thehydrophobic membrane filter 117 as the second layer used was a Mitexmembrane filter (Nihon Millipore Ltd.) having a pore size of 10 μm.

The other container was a container 1002 comprising only a single-layerhydrophilic membrane filter 1001 as the hydrophilic membrane filter(FIG. 14B-a). The single-layer hydrophilic membrane filter 1001 used wasan MF-Millipore membrane filter (Nihon Millipore Ltd.) having a poresize of 0.45 μm, a thickness of 150 μm, and porosity of 79% and wasprocessed into 0.5 cm in diameter.

200 μL of 0.2% benzalkonium chloride+25 mM Tricine buffer, pH 12 wasadded as a microbial dissolution solution 703 onto each of the two-layermembrane filter 101 and the single-layer hydrophilic membrane filter1001 and left standing for 10 minutes. After the 5- and 10-minutestanding, no change was observed in the two-layer membrane filter 101(FIGS. 14A-b and 14A-c). By contrast, the microbial dissolution solution1003 penetrated through the single-layer hydrophilic membrane filter1001 and dropped off, with standing time (FIGS. 14B-b and 14B-c).

For measuring the amount of the microbial dissolution solution 1003 thatpenetrated through the single-layer hydrophilic membrane filter 1001 anddropped off, weight measurement was attempted by collecting themicrobial dissolution solution 1003 that penetrated through the membranefilter and dropped off. 4 minutes after the addition of the microbialdissolution solution 703, 10 mg of the microbial dissolution solution1003 penetrated through the membrane filter and dropped off. Inmeasurement at 6 and 8 minutes thereafter, 15 mg and 20 mg of themicrobial dissolution solution 1003, respectively, penetrated throughthe membrane filter and dropped off. A total of 45 mg of the microbialdissolution solution 1003 was found to penetrate through the membranefilter and drop off.

On the other hand, the microbial dissolution solution neither penetratedthrough the two-layer membrane filter 101 nor dropped off. Instead, thetotal weight of the container 102 comprising the two-layer membranefilter 101 and the microbial dissolution solution 703 was measuredimmediately after the addition of the microbial dissolution solution 703onto the two-layer membrane filter 101 (FIG. 14A-a) and after 10-minutestanding (FIG. 14A-c), and the difference therebetween was calculated.The difference was 1 mg, demonstrating almost no change in the weight.This result indicates that the microbial dissolution solution isretained in the container 102 comprising the two-layer membrane filter.

This result demonstrated that the two-layer membrane filter can retainthereon the microbial dissolution solution.

Experimental Example 3

(Elution Experiment on ATP within Membrane Filter)

The amount of ATP molecules eluted from within the membrane filter wascompared between the two-layer membrane filter and a single-layerhydrophilic membrane filter. For this purpose, two containers comprisingeither membrane filter were prepared. One of the containers was thecontainer 102 comprising the two-layer membrane filter 101 in the bottom(FIG. 15A). The two-layer membrane filter 101 comprised a first layer asan upper layer serving as a hydrophilic membrane filter 116 and a secondlayer as a lower layer serving as a hydrophobic membrane filter 117. Thehydrophilic membrane filter 116 as the first layer used was anMF-Millipore membrane filter (Nihon Millipore Ltd.) having a pore sizeof 0.45 μm, a thickness of 150 μm, and porosity of 79% and was processedinto 0.5 cm in diameter. The hydrophobic membrane filter 117 as thesecond layer used was a Mitex membrane filter (Nihon Millipore Ltd.)having a pore size of 10 μm and was processed into 0.5 cm in diameter.

The other container was a container 1002 comprising only a single-layerhydrophilic membrane filter 1001 as the hydrophilic membrane filter inthe bottom (FIG. 16A). The single-layer hydrophilic membrane filter 1001used was an MF-Millipore membrane filter (Nihon Millipore Ltd.) having apore size of 0.45 μm, a thickness of 150 μm, and porosity of 79% and wasprocessed into 0.5 cm in diameter.

Lucifer ATP Standard Reagent (Kikkoman Corp.) was used as an ATPsolution to prepare an ATP solution having a concentration of 20000amol/10 mL. A luminescent reagent included in Lucifer HS Set (KikkomanCorp.) for causing ATP/luciferase/luciferin reaction was used as aluminescent reagent 115.

First, 10 μL of the ATP solution was added onto each of the two-layermembrane filter 101 and the single-layer hydrophilic membrane filter1001 to infiltrate ATP molecules 1501 into each of the hydrophilicmembrane filter 116 as the first layer and the single-layer hydrophilicmembrane filter 1001 (FIGS. 15A and 16A). Subsequently, 200 μL of 0.2%benzalkonium chloride+25 mM Tricine buffer, pH 12 was added thereonto asa microbial dissolution solution 703 and then left standing (FIGS. 15Band 16B).

While left standing, the microbial dissolution solution 1003 penetratedthrough the single-layer hydrophilic membrane filter 1001 and droppedoff, with time (FIGS. 16C and 16D). On the other hand, no change wasobserved in the two-layer membrane filter 101 (FIGS. 15C and 15D).

For quantitatively evaluating the number of ATP molecules 1601 in themicrobial dissolution solution 1003 that penetrated through thesingle-layer hydrophilic membrane filter 1001 and dropped off as well asthe number of ATP molecules 1501 liberated into the microbialdissolution solution 703 on the single-layer hydrophilic membrane filter1001 (FIGS. 16C and 16D), a 10 μL aliquot was collected from each of themicrobial dissolution solution 703 remaining on the single-layerhydrophilic membrane filter 1001 and the microbial dissolution solution1003 that penetrated through the single-layer hydrophilic membranefilter 1001 and dropped off. The collected solution was added to theluminescent reagent 115 in the reaction container 113, and luminescencegenerated through the reaction was detected by the detection portion112.

4, 6, and 8 minutes after the addition of the microbial dissolutionsolution 703 onto the single-layer hydrophilic membrane filter 1001, themicrobial dissolution solution 1003 that penetrated through the membranefilter and dropped off exhibited luminescence intensity of approximately20000 CPS (count per second), 25000 CPS, and 30000 CPS, respectively(FIG. 17). 20000 amol of ATP molecules existed in the single-layerhydrophilic membrane filter 1001 before penetrating through the membranefilter and dropping off, and 200 μm of the microbial dissolutionsolution 703 was added onto the single-layer hydrophilic membrane filter1001. Taking this into consideration, the ATP concentration isapproximately 1000 amol/10 μL when the ATP molecules 1501 are ideallydistributed into the microbial dissolution solution 703. Luminescenceintensity at the ATP concentration of 1000 amol/10 μL is determined inmeasurement to be approximately 10000 CPS. Thus, the number of ATPmolecules 1601 in the microbial dissolution solution 1003 thatpenetrated through the single-layer hydrophilic membrane filter 1001 anddropped off was found to be 2 to 3 times larger than that obtained bythe ideal distribution of ATP molecules 1501 into the microbialdissolution solution 703.

On the other hand, the number of ATP molecules 1501 contained in themicrobial dissolution solution 703 remaining on the single-layerhydrophilic membrane filter 1001 was measured based on luminescence. Asa result, approximately 5000 CPS was the maximum even after 10-minutestanding. This luminescence intensity was ¼ to ⅙ of that measured fromthe microbial dissolution solution 1003 that penetrated through themembrane filter and dropped off (FIG. 17). This result demonstrated thatonly approximately ¼ to ⅙ of the ATP molecules 1501 (FIG. 16A) withinthe single-layer hydrophilic membrane filter 1001 is liberated into thesolution on the hydrophilic membrane filter 1001 (FIG. 16D).

On the other hand, the microbial dissolution solution 703 neitherpenetrated through the two-layer membrane filter 101 nor dropped off.Instead, a 10 μL aliquot was collected from the microbial dissolutionsolution 703 on the two-layer membrane filter 101 immediately after theaddition of the microbial dissolution solution 703 onto the two-layermembrane filter 101 and after 2- to 20-minute standing. The collectedsolution was subjected to luminescence measurement. At each of thestanding times, luminescence intensity was shown to be approximately9800 CPS (FIG. 18). This result indicates that the ATP molecules 1501(FIG. 15A) infiltrated in the hydrophilic membrane filter 116 as thefirst layer can be eluted into the solution on the two-layer membranefilter 101 by retaining the microbial dissolution solution 703 in thecontainer 102 comprising the two-layer membrane filter 101 (FIG. 15D).

This result demonstrated that the ATP molecules 1501 can be elutedefficiently from within the hydrophilic membrane filter 116 as the firstlayer by retaining the microbial dissolution solution 703 on thetwo-layer membrane filter 101. The elution can be performed moreefficiently by use of the hydrophilic membrane filter as the first layerhaving a smaller diameter and a smaller thickness.

Experimental Example 4

(Experiment on Measurement of Microbes Using Wetting Agent)

Microbial detection sensitivity was compared between the two-layermembrane filter and a hydrophobic membrane filter capable of filteringan aqueous solution by virtue of a wetting agent infiltrated therein.For this purpose, four containers comprising a membrane filter wereprepared.

(1) Container Comprising Two-Layer Membrane Filter:

The two-layer membrane filter comprised a first layer as an upper layerserving as a hydrophilic membrane filter and a second layer as a lowerlayer serving as a hydrophobic membrane filter. The hydrophilic membranefilter as the first layer used was an MF-Millipore membrane filter(Nihon Millipore Ltd.) having a pore size of 0.45 μm, a thickness of 150μm, and porosity of 79% and was processed into 0.5 cm in diameter. Thehydrophobic membrane filter as the second layer used was a Mitexmembrane filter (Nihon Millipore Ltd.) having a pore size of 10 μm andwas processed into 0.5 cm in diameter.

(2) Container Comprising Single-Layer Hydrophilic Membrane Filter:

The single-layer hydrophilic membrane filter used was an MF-Milliporemembrane filter (Nihon Millipore Ltd.) having a pore size of 0.45 μm, athickness of 150 μm, and porosity of 79% and was processed into 0.5 cmin diameter.

(3) Container Comprising Wetting Agent-Requiring Two-Layer MembraneFilter which Comprises Hydrophilic Membrane Filter as First Layer andHydrophobic Membrane Filter Capable of Filtering Aqueous Solution byVirtue of Wetting Agent Infiltrated Therein, as Second Layer:

The hydrophilic membrane filter as the first layer used in the two-layermembrane filter was an MF-Millipore membrane filter (Nihon MilliporeLtd.) having a pore size of 0.45 μm, a thickness of 150 μm, and porosityof 79% and was processed into 0.5 cm in diameter. The hydrophobicmembrane filter as the second layer used was a hydrophobic Duraporemembrane filter (Nihon Millipore Ltd.) having a pore size of 0.45 μm andwas processed into 0.5 cm in diameter.

(4) Container Comprising Wetting Agent-Requiring Single-LayerHydrophobic Membrane Filter Which Comprises Hydrophobic Membrane FilterCapable of Filtering Aqueous Solution by Virtue of Wetting AgentInfiltrated Therein:

The single-layer hydrophobic membrane filter used was a hydrophobicDurapore membrane filter (Nihon Millipore Ltd.) having a pore size of0.45 μm and was processed into 0.5 cm in diameter.

E. coli was used as the microbe to be measured. E. coli was suspended ina phosphate buffer (pH 7.4) (Invitrogen Corp.) to prepare an E. colisuspension having 100 individuals of E. coli/10 mL. An ATP eliminationsolution included in the Lucifer HS Set (Kikkoman Corp.) was used as anextra-microbial biological material removal reagent. An ATP extractionsolution included in the Lucifer HS Set (Kikkoman Corp.) was used as amicrobial dissolution solution.

10 mL of the E. coli suspension was added onto each of the two-layermembrane filter, the single-layer hydrophilic membrane filter, thewetting agent-requiring two-layer membrane filter, and the wettingagent-requiring single-layer hydrophobic membrane filter. Then, 10 μm ofthe ATP elimination solution was added thereto as a first reagent.

The two-layer membrane filter and the single-layer hydrophilic membranefilter could filter the E. coli suspension. However, the wettingagent-requiring two-layer membrane filter and the wettingagent-requiring single-layer hydrophobic membrane filter failed tofilter the E. coli suspension. Therefore, each hydrophobic membranefilter was infiltrated with methyl alcohol as a wetting agent to achievefiltration.

After the filtration, 200 μL of the ATP extraction solution was addedthereto as a second reagent. 10 μL of the ATP extraction solutionremaining on each of the two-layer membrane filter, the single-layerhydrophilic membrane filter, the wetting agent-requiring two-layermembrane filter, and the wetting agent-requiring single-layerhydrophobic membrane filter was subjected to luminescence measurement.

For the two-layer membrane filter, 140 amol of ATP was detected from 100individuals of E. coli. On the other hand, for the single-layerhydrophilic membrane filter, 10 amol ATP was detected, whereas for thewetting agent-requiring two-layer membrane filter and the wettingagent-requiring single-layer hydrophobic membrane filter, 12 amol of ATPwas detected. The number of ATP detected using each membrane filter wasapproximately 1/10 or less of that detected using the two-layer membranefilter.

This result may be because E. coli-derived ATP flowed out duringfiltration due to damage or dissolution of E. coli by the wetting agentinfiltrated in the hydrophobic membrane filter.

The microbial detection method which involves filtration through ahydrophobic membrane filter using a wetting agent reduces both microbialdetection sensitivity and precision. On the other hand, the two-layermembrane filter without the use of a wetting agent improved microbialdetection sensitivity and precision.

DESCRIPTION OF SYMBOLS

-   101 two-layer membrane filter-   102 container-   103 reagent supply portion-   104 pipe for reagent supply-   105 suction portion-   106 washing solution supply portion-   107 pipe for washing solution supply-   108 heating portion-   109 arm-   110 dispensing portion-   111 pipe for dispensing-   112 detection portion-   113 reaction container-   114 input/control portion-   115 luminescent reagent-   116 hydrophilic membrane filter-   117 hydrophobic membrane filter-   118 waste liquid portion-   301 reagent-   302 reagent reservoir-   601 excitation light irradiation portion-   701 E. coli-   702 E. coli suspension-   703 microbial dissolution solution-   704 E. coli-derived ATP molecules-   801 bottom of container-   802 annular cap-   803 screw-   901 screwed bottom of container-   902 screwed annular cap-   1001 single-layer hydrophilic membrane filter-   1002 container-   1003 microbial dissolution solution that penetrated through membrane    filter and dropped off-   1004 E. coli-derived ATP molecules that penetrated through membrane    filter and dropped off-   1101 suction port-   1501 ATP molecules-   1601 ATP molecules that penetrated through membrane filter and    dropped off

What is claimed is:
 1. A sample container for a microbial detection process comprising: a two-layer membrane filter comprising a hydrophilic membrane filter as an upper layer, into which a solution is supplied, and a hydrophobic membrane filter as an underlying lower layer, into which the solution having passed through the hydrophilic membrane filter is supplied; and a container which holds the two-layer membrane filter in a bottom portion where the outer edge of the two-layer membrane filter is secured, wherein the hydrophilic membrane filter has a pore size smaller than that of the hydrophobic membrane filter, wherein the hydrophobic membrane filter is porous membrane filter having a power size of 0.8 μm to 80 μm or a membrane filter having a mesh structure with a pitch size of 1 μm to 59 μm, and wherein the hydrophilic membrane filter has a pore size of 0.05 μm to 0.65 μm.
 2. The sample container for the microbial detection process according to claim 1, wherein the pore size of the hydrophilic membrane filter is sized to capture a microbe, and the pore size or pitch size of the hydrophobic membrane filter is sized to retain a reagent.
 3. The sample container for the microbial detection process according to claim 1, wherein the hydrophobic membrane filter has a thickness of 7 μm to 800 μm.
 4. The sample container for the microbial detection process according to claim 1, wherein the hydrophilic membrane filter has a thickness of 7 μm to 200 μm.
 5. The sample container for the microbial detection process according to claim 1, wherein when an aqueous solution is contained in the sample container, and being present above the two layer membrane filter, the aqueous solution can be filtered through the hydrophilic membrane filter and the hydrophobic membrane filter by application of a negative pressure below the hydrophobic membrane filter.
 6. A microbial detection apparatus comprising: a sample container comprising, in a bottom portion thereof, a two-layer membrane filter comprising a hydrophilic membrane filter as an upper layer, into which a solution is supplied, and a hydrophobic membrane filter as an underlying lower layer, into which the solution having passed through the hydrophilic membrane filter is supplied; a suction portion which forms a negative pressure below the two-layer membrane filter; a reagent supply portion which separately adds an extra-microbial biological material removal reagent and a microbial dissolution solution to the sample container; a detection portion which detects luminescence from a sample; and a control portion which controls each portion in the apparatus, the control portion being configured to actuate the reagent supply portion to add the extra-microbial biological material removal reagent to a sample in the sample container, then actuate the suction portion to filter the solution in the sample container through the two-layer membrane filter, and then actuate the reagent supply portion to add the microbial dissolution solution to the sample container, wherein the hydrophilic membrane filter has a pore size smaller than that of the hydrophobic membrane filter, wherein the hydrophobic membrane filter is porous membrane filter having a power size of 0.8 μm to 80 μm or a membrane filter having a mesh structure with a pitch size of 1 μm to 59 μm, and wherein the hydrophilic membrane filter has a pore size of 0.05 μm to 0.65 μm.
 7. The microbial detection apparatus according to claim 6, wherein the reagent supply portion supplies a luminescent reagent to the sample container, and the detection portion detects luminescence generated from the solution in the sample container thus supplied with the luminescent reagent.
 8. The microbial detection apparatus according to claim 6, further comprising a dispensing portion, wherein the dispensing portion dispenses the microbial dissolution solution to a reaction container, and the detection portion detects luminescence generated through the reaction between the sample solution thus dispensed with the microbial dissolution solution and a luminescent reagent.
 9. The microbial detection apparatus according to claim 6, further comprising an excitation light irradiation portion, wherein the detection portion detects fluorescence generated from the sample by excitation light irradiation from the excitation light irradiation portion. 